20 Apoptosis in the Physiology and Diseases of the Respiratory Tract

The lung provides a huge contact interface between the organism and its environment. Its mucosal surfaces must permit gas exchange between the blood and air, but also act as a barrier against a plethora of microorganisms. In addition, inhaled toxins and particles may enter the organism via the lung. Accordingly, inflammatory airway and lung diseases are among the most prevalent human morbidities. Lung cancer, which in most cases can be attributed to tobacco smoking, is the leading cause of cancer-related mortality in the developed world. In this chapter, we summarize the role of apoptotic cell death in lung development and in clinical disease states.

1. APOPTOSIS IN LUNG DEVELOPMENT

The physiology and genetics of the branching program underlying lung development are just being unraveled. Following the initial separation of the lung bud from the prospective esophagus in the embryonic stage, apoptosis of mesenchymal and epithelial cells can be observed during the various stages of lung development. Regulation of developmental apoptosis has been linked to the expression of cytokines, such as transforming growth factor-β1 (TGF-β1) and insulin-like growth factor 1 (IGF-1), as well as additional apoptosis-related proteins and nitric oxide. In the early stages of lung development, apoptosis is mainly detectable in the mesenchymal tissue layer. Indeed, apoptosis was almost exclusively found in the regions of new bud formation or in the mesenchyme underlying branch points, thus providing space for the outgrowth of lung buds. During later stages of lung development, both alveolar epithelial and mesenchymal tissue apoptosis can be detected. At this time, apoptosis coincides with airway branching, decreased cell proliferation, and alveolar epithelial thin-

ning, thus implying cellular apoptosis as a significant contributor of lung remodeling. Alveolarization and microvascular maturation do not stop at birth, but continue up to a few years after birth(1). After birth, apoptosis emerges as an important process after extensive proliferation of type 2 alveolar epithelial cells, which are produced in higher numbers than actually required. Consequently, type 2 cells are removed either by differentiation or by apoptosis, thus preserving functional alveoli.

Using different approaches, genes encoding most of the core factors of the apoptotic machinery have been targeted in the mouse. Although some of these genetargeted mice exhibited developmental defects, none of them showed particular pathology in the respiratory tract or lung. This observation does not rule out an involvement of apoptosis regulators in lung physiology, as some of these knockout mice, such as those deficient in Mcl-1, cytochrome c, caspase-8, casper, or FADD, succumb during embryonic development, and their lung development cannot be examined. Recently, mice with targeted deletion of the miR-17 92 microRNA were described to exhibit embryonic lethality due to lung hypoplasia and cardiac defects. Examination of lung tissues revealed increased expression of the proapoptotic BH3-only protein Bim, and miR-17 92 was found to transcriptionally repress Bim. Overall, these findings suggest that apoptosis plays an important role in mammalian lung development.

2. APOPTOSIS IN LUNG PATHOPHYSIOLOGY

2.1. Apoptosis in pulmonary inflammation

The lung is characterized by a large mucosal surface at risk for exposure to many types of microorganisms (e.g., viral, bacterial) and irritants (e.g., ozone), which

can lead sometimes to severe inflammatory reactions. Apoptotic cells are usually rapidly and effectively cleared from the lung so that no apoptotic cells are detectable in the healthy lung. Even during extensive inflammatory reactions, (e.g., during lung infection), few apoptotic cells are found in lung tissue. Neutrophils derived from broncho-alveolar lavage (BAL) samples of patients with pneumonia even display decreased rates of apoptosis. Also, during resolution of lung inflammation, apoptotic cells are rapidly removed by macrophages through cell recognition receptors (phosphatidylserine receptor, CD36, and alpha v integrins). Uptake of apoptotic cells by macrophages then leads to a noninflammatory environment by production of anti-inflammatory mediators that include TGF-β, interleukin-10 (IL-10), and prostaglandin E2 (PGE2). Indeed, in animal studies, instillation of apoptotic cells into the lung results in rapid resolution of inflammatory responses, suggesting that apoptosis and uptake of apoptotic cells provides an intrinsic anti-inflammatory circuit that attenuates proinflammatory responses. However, in several lung diseases, increased numbers of apoptotic cells have been described in the airways and lung tissue, implying either an increase in apoptosis or defects in clearance of apoptotic cells in pathophysiology. In cystic fibrosis, which is characterized by a massive influx of inflammatory cells and release of proteases in the lung, increased numbers of apoptotic neutrophils are detectable in the airway. This finding has been linked to cleavage of the phosphatidylserine receptor on macrophages by neutrophil elastase, which impairs the uptake of apoptotic cells and contributes to ongoing inflammation. Similarly, alveolar macrophages from patients with chronic obstructive pulmonary disease (COPD) are less effective in phagocytosing apoptotic airway epithelial cells as compared with controls. Also, increased apoptosis of lung structural cells has been described in several lung diseases, including acute lung injury, COPD, and lung fibrosis.

2.2. Apoptosis in acute lung injury

Acute lung injury (ALI) and the more severe acute respiratory distress syndrome (ARDS) represent clinical syndromes that result from complex responses of the lung to a multitude of direct and indirect stresses. Important pathophysiologic changes found in patients with ALI are alveolar inflammation and injury. Indeed, epithelial injury is one of the hallmarks of ALI in patients, and alveolar epithelial cells are especially affected. Similar to patients with pneumonia, only few apoptotic inflammatory cells are found in the lung, probably due to increases

in growth factors and rapid uptake of apoptotic granulocytes by macrophages. In contrast, it has recently become clear that increased apoptosis is detectable in parenchymal lung cells of adult patients with ALI and ARDS. Similar observations have also been reported in newborns with acute lung injury. Especially alveolar epithelia cells have been found to be apoptotic, leading to increased epithelial permeability and subsequent alveolar flooding. The increase in alveolar cell apoptosis could be mediated by increased levels of soluble Fas ligand (sFasL), which can be detected in BAL samples of patients with ALI and ARDS. During onset of ARDS, sFasL is highly biologically active and induces apoptosis of alveolar epithelial cells in vitro, particularly affecting distal epithelia cells. Also, in patients with ARDS, Fas expression on alveolar epithelial cells that line the alveolar walls is increased. Together, these findings are strongly suggestive that activation of the Fas pathway is an important contributor to alveolar epithelial cell apoptosis leading to the development of ALI and ARDS, in addition to other factors such as mechanical stress, hyperoxia, and hypoxia.

Animals models of ALI have confirmed that apoptosis of parenchymal lung cells contributes to acute lung damage. Indeed, direct instillation of sFasL into the lung or administration of an activating anti-Fas antibody leads to increased alveolar cell apoptosis associated with increased pulmonary inflammation. Also, meconium instillation into the lung, as a model for acute lung injury in newborns, leads to increased apoptosis of epithelial cells. Additionally, models of lung hyperoxia have revealed that apoptosis is a prominent component of the acute response, which is also mediated by Fas/FasL pathway. Other models of ALI involve instillation of bacterial lipopolysaccharides (LPS) into the lung. In these models, increased apoptosis of alveolar epithelial cells and interstitial inflammatory cells are also detectable. In addition to epithelial cell apoptosis, endothelial cell apoptosis can be found in models of hemorrhagic shock. Interestingly, administration of blocking anti-Fas antibodies or caspase-3 inhibitors attenuates lung injury after LPS instillation, suggesting that regulation of apoptosis could be a potential treatment of ARDS.

However, apoptosis also has beneficial effects during the resolution of inflammation after acute lung injury. Indeed, during resolution of ALI, hyperplasia of type II pneumocytes occurs as a reparative phenomenon. During clearance of inflammation, extensive apoptosis of type II pneumocytes mediated by Fas/FasL accounts for the disappearance of these cells from the lung. Therefore, although blocking apoptosis at an early stage of the disease might be beneficial for the course of ALI

and ARDS, inhibiting apoptosis at later stages might be counter-indicated because apoptosis and engulfment of apoptotic cells are important events during resolution of inflammation.

2.3. Apoptosis in chronic obstructive pulmonary disease

COPD is a chronic respiratory disease characterized by airflow limitation that is not fully reversible. The airflow limitation usually is progressive and associated with inflammation of the lungs. COPD is a combination of two phenotypes, chronic obstructive bronchitis, defined clinically as chronic productive cough in combination with airflow obstruction, and emphysema, characterized pathologically as the presence of permanent enlargement of the airspaces distal to the terminal bronchioles, accompanied by destruction of their walls. COPD is the result of exposure to noxious particles. Cigarette smoke is the major risk factor for the development of this disease in the Western world. However, despite progress in the characterization of COPD, so far the pathophysiologic cause has not been identified. Several mechanisms have been suggested to contribute to the development of COPD. Airway inflammation, oxidative stress, a disrupted balance between proteolytic and antiproteolytic molecules in the lung, as well as premature aging and senescence are suspected to contribute to the development of COPD. In recent years, alternative mechanisms have been discussed. There is increasing evidence from studies in humans as well as data from animal models that defective homeostasis of apoptosis and proliferation in the lung might lead to a disruption of alveolar architecture, leading to emphysema.

Indeed, several studies in lung tissue samples from patients with COPD have described increased apoptosis as compared with controls. Increased apoptosis was found in several types of lung cells, including endothelial cells, interstitial cells, and alveolar epithelial cells. In addition, increased detection of active caspase-3 and expression of Bax and Bad as well as elevated levels of airway granzyme B and perforin have been reported, providing evidence for enhanced activation of proapoptotic pathways in these patients.

Animal models of COPD have also suggested an important role of apoptosis in the development of emphysema, even in the absence of significant inflammatory changes. Experimental observations, which describe alveolar enlargement as a result of alveolar and endothelial cell apoptosis, have supported the concept of a direct involvement of apoptosis in emphysema development. Direct targeting of alveolar cells in mice by intratracheal

administration of active caspase-3 or ceramide results in epithelial apoptosis and development of emphysematous changes in mice. Inhibition of growth factor signaling in the lung also results in increased apoptosis. Indeed, targeting vascular endothelial growth factor (VEGF) or VEGF receptor (VEGFR) in the lung increases apoptosis of alveolar cells, leading to enlargement of the alveolar space. This process can be attenuated by treatment with a caspase inhibitor, thus preventing apoptosis and development of emphysema. Similar to animal studies, decreased expression of VEGF and VEGFR has been detected in patients with emphysema, suggesting that epithelial and endothelial alveolar septal death due to a decrease of endothelial cell maintenance factors contributes to the pathogenesis of emphysema. Some studies have linked increased apoptosis with other pathophysiologic mechanisms of emphysema development (such as protease/antiprotease imbalance). Mice that over-express interferon γ in the lungs develop emphysema and have increased numbers of apoptotic parenchymal lung cells. Interestingly, inhibition of cathepsin S, an important elastase in the lung, results in decreased apoptosis and emphysema in interferon-expressing transgenic mice. This suggests that protease-dependent epithelial cell apoptosis is a critical event in the pathogenesis of alveolar remodeling and emphysema, linking apoptosis to protease/antiprotease imbalance. Overall, these findings show that apoptosis of lung cells plays an important role during the development of COPD and especially emphysema. Thus far, no therapeutic approaches have been clinically developed to prevent apoptosis in this patient group.

2.4. Apoptosis in interstitial lung diseases

Interstitial lung diseases constitute a heterogeneous collection of diseases that are characterized by a progressive distortion of the alveolar architecture and replacement by fibrotic tissue. The clinically most relevant form is idiopathic pulmonary fibrosis (IPF), which is characterized by progressive dyspnea, decline in lung function, and death within 3 to 4 years from diagnosis. Histopathologically, IPF typically shows a pattern of usual interstitial pneumonia, the cardinal features of which are patchy fibrosis with variable numbers of fi- broblastic foci interspersed with areas of normal or nearly normal lung. Although initial studies focused on the role of inflammation in inciting fibroblast activation and fibrosis, current concepts suggest a central role of the epithelium in IPF pathogenesis. It has been proposed that IPF is the result of ongoing alveolar epithelial

injury and subsequent dysregulated repair associated with the formation of fibroblast-myofibroblast foci, which evolve into fibrosis. A relatively small increase in the rate of epithelial cell apoptosis is predicted to result in considerable cell loss over time and thus minor upregulation of epithelial apoptosis, especially of alveolar epithelial cells, could account for excessive epithelial loss. Therefore, it is likely that apoptosis of epithelial cells is important in the injury and repair processes present in IPF. Indeed, alveolar epithelial cell injury and apoptosis were found in the lungs of patients with IPF, even in areas that histologically seemed normal. Especially in areas where epithelial cells are in close proximity to myofibroblasts, increased epithelial cell apoptosis is frequently detectable. In addition, lung tissues from IPF patients exhibit increased expression of proapoptotic proteins (p53, Bax, and caspase-3) and decreased expression of antiapoptotic proteins (Bcl-2) in epithelial cells.

Animal studies have also demonstrated that apoptosis of alveolar epithelial cells is sufficient to induce pulmonary fibrosis. Intratracheal instillation of activating anti-Fas antibody induces apoptosis of alveolar epithelial cells and results in pulmonary fibrosis in rodents. Apoptosis is also induced by over-expression of TGF- β in the rodent lung, leading to inflammation, myofibroblast hyperplasia, tissue fibrosis, and honeycombing. Treatment with caspase inhibitors markedly ameliorates fibrosis and alveolar remodelling. In addition, apoptosis of alveolar epithelial cells is detected. In the commonly used bleomycin-induced pulmonary fibrosis model, direct inhibition of apoptosis by blocking the Fas/FasL pathway results in a blunted apoptotic response to bleomycin and decreased collagen production. Also, treatment with caspase inhibitors not only prevents apoptosis, but also reduces the histopathological grade of lung inflammation and decreases fibrosis.

2.5. Apoptosis in pulmonary arterial hypertension

Pulmonary hypertension (PH) is a hemodynamic and pathophysiological condition defined as an increase in pulmonary artery pressure ≥25 mmHg at rest as assessed by right heart catheterization. Pulmonary arterial hypertension (PAH) is a clinical condition characterized by the presence of pre-capillary PH in the absence of other causes of pre-capillary PH such as PH due to lung diseases, chronic thromboembolic PH, or other rare diseases. PAH includes different forms that share a similar clinical picture and virtually identical pathological changes like vasoconstriction, in situ thrombosis, and vascular remodeling of pulmonary arteries (Gaile et al. 2009).

PAH can be classified into five categories: (1) idiopathic PAH, (2) heritable PAH, (3) drugor toxin-induced PAH, (4) PAH associated with other diseases (e.g. connective tissue diseases, HIV infection, portal hypertension), and (5) persistent pulmonary hypertension of the newborn. PAH is a progressive disease characterized by abnormal muscularization of distal pulmonary arteries, striking reduction in arterial numbers, progressive intimal hyperplasia leading to occlusive changes in the pulmonary arteries, and so-called plexiform lesions. The initial pathological events are thought to be related to dysregulation of pulmonary artery smooth muscle proliferation. Increased proliferation and decreased apoptosis of pulmonary arterial smooth muscle could mediate thickening of the pulmonary vasculature, which subsequently would lead to reduced inner diameter and increased pulmonary vascular resistance. Several lines of evidence have suggested an impaired regulation of pulmonary artery smooth muscle proliferation. In a subset of PAH patients, loss-of-function mutations in bone morphogenetic protein receptor 2 (BMPR2) has been found. Activation of the BMPR2 pathway results normally in suppression of arterial smooth muscle cell proliferation. In contrast, arterial smooth muscle cells from patients with PAH were not inhibited in their proliferation after BMPR2 activation. Also, mediators that favor suppression of apoptosis (e.g., Bcl-2) are upregulated in lung vessels of patients with PAH. These findings suggest that some abnormalities described in PAH contribute to resistance to apoptosis and a proliferation/apoptosis imbalance within the vascular wall, thus leading to smooth muscle proliferation and vascular remodeling. These hypotheses are supported by the description of increased expression of the Survivin protein in remodeled pulmonary arteries from patients with PAH. In this regard, dysregulated Survivin expression is considered to be a major pathological mechanism in animal models of PAH, which have demonstrated Survivin over-expression coinciding with pulmonary vascular remodeling. Furthermore, inhibition of Survivin by gene therapy in these models resulted in pulmonary artery smooth muscle cell apoptosis and decreased pulmonary vascular resistance, heart failure, and vascular remodeling. Theses finding suggest that targeted pro-apoptotic agents could be a possible new therapeutic approach for patients with PAH.

2.6. Apoptosis in lung cancer

Lung cancer is the leading cause of cancer mortality in the United States and Western Europe. The main risk factor for the development of lung cancer is inhaled tobacco exposure. Smoking 20 cigarettes per day for

20 years (i.e., 20 pack-years) increases the age-adjusted risk for lung cancer approximately 20-fold. In the light of the high prevalence of tobacco smoking in Asia, Eastern Europe, Latin America, and South America, lung cancer seems destined to become an even larger global health problem, with enormous socioeconomic and health care costs. Based on histology and clinical course, lung cancers have been grouped into small-cell lung cancer (SCLC), which comprises less than 20% of lung cancer cases, virtually all of which are associated with cigarette smoking, and non–small-cell lung cancers (NSCLC), which constitute more than 80% of lung cancer cases. The latter is a heterogeneous group composed of several histologies, such as squamous cell carcinoma (SCC), adenocarcinoma, large-cell carcinoma, bronchoalveolar carcinoma, and others. Historically, all NSCLCs have been uniformly treated. However, more recently it has been recognized that some NSCLC subgroups are more susceptible to certain therapies. For example, patients with adenocarcinoma and no smoking history have a high incidence of amplification and mutations of the epidermal growth factor receptor (EGFR), which make them prone to responding to EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib or gefitinib. Also, the antifolate pemetrexed achieved superior survival outcomes in patients with adenocarcinoma and large-cell carcinoma, but not those with SCC. Further, SCC patients are excluded from receiving the anti-VEGF antibody bevacizumab in combination with chemotherapy because of a higher risk of bleeding complications. These clinical and histological distinctions are currently substantiated by more sophisticated efforts of tumor characterization, such as gene expression profiling and massively parallel sequencing. Hence it is expected that molecular predictors and specific targets will play an even larger role in future treatment decisions for patients with lung cancer.

Against this background, the analysis of apoptosis pathways in lung cancer is of particular importance. First, as in most cancers, deregulation of apoptosis is an important event in lung carcinogenesis. This is exemplified by inactivating mutations of the TP53 tumor suppressor gene, which are found in approximately half of lung cancers. Accordingly, loss of p53 accelerates tumor development in a mouse model of K-ras–induced lung carcinogenesis. p53 gene transfer and pharmacological restoration of p53 function have been shown to induce apoptosis in p53-defective lung cancer cells in vitro and in pilot studies of somatic gene therapy in vivo. Additional genes involved in apoptosis regulation were found to be differentially expressed in murine lung cancer models as well as in human lung cancer samples as compared with nonmalignant tissues. In addition,

hypothesis-driven studies have specifically addressed apoptosis regulators with known function. For example, in a transgenic model of Raf-induced lung carcinogenesis, the onset of tumor development was greatly delayed by targeted deletion of Bcl-2. Accordingly, protein expression patterns of various proand antiapoptotic Bcl-2 family proteins correlated with prognosis in surgically resected lung cancer patients in some studies, in addition to changes in caspase expression. Taken together, these studies support a role of apoptosis in the development and progression of lung cancer. Hence apoptosis-directed strategies merit examination for lung cancer prevention and treatment. Indeed, at present, a chemical inhibitor of antiapoptotic Bcl-2 family proteins (BH3 mimetic) is currently in clinical testing for SCLC. Prospective studies are needed to define a role for expression analysis of apoptosis regulators as prognostic factors or predictors for treatment decisions in lung cancer patients.

Second, cytotoxic chemotherapy and gamma radiation, which are both thought to exert at least some of their activities by inducing apoptosis, still provide the basis of current standard treatment protocols for patients with nonresectable advanced and metastatic lung cancers. In addition, EGFR mutations in lung cancer were shown to activate antiapoptotic pathways, and this was reversed by EGFR TKI treatment. Hence deregulation in apoptotic signal transduction could interfere with the therapeutic activity of lung cancer therapies. Alternatively, proapoptotic therapies could overcome resistance to current drugs in clinical use for NSCLC and SCLC patients. Expression of antiapoptotic Bcl-2 family proteins was found to interfere with drug sensitivity of cancer cells, which is overcome by gene transfer-mediated expression of proapoptotic Bcl-2 proteins. These studies provide a lead for the development of pharmacological compounds targeting anti-apoptotic Bcl-2 proteins in lung cancer. Of particular interest are so-called BH3 mimetics, which exhibit promising activity in preclinical models of SCLC and NSCLC. Additional apoptotic targets have been identified that could enhance the efficacy of cytotoxic lung cancer therapies. The mitochondrial protein Smac is thought to interfere with the inhibition of caspases by inhibitor of apoptosis (IAP) proteins. Accordingly, Smac-derived peptides were shown to sensitize lung cancer cells to cytotoxic anticancer drugs in vitro. However, recent studies with smallmolecule IAP inhibitors suggest a role in tumor necrosis factor (TNF) signaling, in addition to allowing caspase activation. Conditional expression of pp32/PHAPI, a putative modulator of apoptosome activity, sensitizes drug-resistant lung cancer cells to apoptosis in vitro and in vivo. Interestingly, high expression of pp32/PHAPI